Lung transplantation is an effective therapy for many end stage lung diseases. Despite advances in immunosuppressive therapy, chronic rejection remains a leading cause of morbidity and mortality. While certain markers of inflammation have been identified currently no set of biomarkers predictably identify patients who are at risk of or currently have chronic rejection. We theorize that there are distinct and reproducible sets of protein products within the lung that characterize chronic rejection and can be characterized into a biosignature of rejection. We chose broncho-alveolar lavage fluid as a starting point to identify a biosignaure of rejection since the small airways are the observable site of chronic rejection and are likely to yield a strong signal. The goal of this project is to identify and validate effective biomarkers for eventual use as diagnostic tools for the detection of chronic lung allograft rejection. This proposal begins with a discovery made during our mass spectrometric analysis of archived bronchoalveolar lavage (BALF) samples of a cohort of well-characterized lung transplant recipients. Samples from patients who subsequently developed chronic rejection or bronchiolitis obliterans syndrome (BOS) had multiple peaks on their mass spectrometry spectrum compared to controls, i.e. those that did not develop BOS. We subsequently identified elevated levels of human neutrophil peptide (HNP) and the decline of Clara Cell Protein as potential biomarkers of BOS. These changes occurred up to 20 months prior to the onset of clinical onset of BOS. In addition, we identified peak ratios by profile analysis of the MALDI-TOF spectrum, including unidentified peaks that predicted those that would develop BOS. Consequently, this proposal focuses on developing diagnostic tools using state-of-the-art proteomics to identify those at risk for developing chronic rejection. We have proposed two major areas of research. One is to validate HNP, Clara Cell Protein and the identified protein ratios as biomarkers of chronic lung transplant rejection in a prospective study of lung transplant recipients. The second is to increase the number of biomarkers for chronic rejection, creating a panel of protein biomarkers that ultimately predicts those with or are at risk for chronic lung transplant rejection.